In bioscience research, medical treatment, drug development and similar fields, it has become increasingly important to comprehensively identify various substances, such as proteins, peptides, nucleic acids and sugar chains. In particular, when aimed at proteins or peptides, such a comprehensive analysis method is called “shotgun proteomics.” For such analyses, the combination of a chromatographic technique, such as a liquid chromatograph (LC) or capillary electrophoresis (CE), with an MSn mass spectrometer (tandem mass spectrometer) has proven itself to be a very powerful technique.
A procedure of a commonly known method for comprehensively identifying various kinds of substances in a biological sample by means of an MSn mass spectrometer is as follows:
[Step 1] Various substances contained in a sample to be analyzed are separated by an appropriate method, e.g. LC or CE. The thereby obtained eluate is preparative-fractionated to prepare a number of small amount samples. (Each of the small amount samples obtained by preparative fractionation is hereinafter called the “fractionated sample.”) In the preparative fractionation of a sample, the sample may be fractionated by various methods: in one method, small amount samples only around peaks are collected; in another method, small amount samples are collected continuously at regular predetermined intervals of time, or small amount samples are constantly collected in the same amount. In any method, it is preferred that every substance in the sample must be included in one of the fractionated samples without fail.
[Step 2] For each fractionated sample, an MSn spectrum is obtained, and a peak or peaks that are likely to have originated from a substance or substances to be identified is selected on the MSn spectrum.
[Step 3] Using the peak selected in Step 2 as the precursor ion, an MS2 analysis is performed on the fractionated sample concerned. Then, based on the result of this analysis, a database search or de novo sequencing is performed to identify a substance contained in the fractionated sample.
[Step 4] If no specific substance has been identified with sufficient accuracy, an MS2 analysis using another peak on the MS1 spectrum as the precursor ion is performed, or a higher-order MSn analysis (i.e. n=3 or greater) using a specific ion observed on the MS2 spectrum as the precursor ion is performed. Then, a database search, de novo sequencing or similar data processing based on the result of the analysis is performed to identify a substance or substances contained in the fractionated sample.
[Step 5] The processes of Steps 2 through 4 are performed for each of the fractionated samples to comprehensively identify various substances contained in the original sample.
To identify each of the substances with high accuracy by the previously described comprehensive identification process, it is desirable that each fractionated sample should contain a small number of kinds of substances (most desirably, only one kind). To achieve this, it is necessary to shorten the period of each fractionating cycle, which significantly increases the number of cycles of fractionation. Considering that, to identify as many substances as possible within a limited period of time, i.e. to improve the throughput of the comprehensive identification of one or more substances contained in a fractionated sample, one or more precursor ions having a higher probability of successful identification (which is hereinafter called the “identification probability”) should be preferentially chosen for the MSn analysis.
One conventional method for selecting a precursor ion for an MS2 analysis from the peaks observed on an MSn spectrum obtained for a given sample is to sequentially select the peaks on the spectrum in descending order of strength (see Patent Document 1). For example, if the length of time for the MS2 analysis of one sample is limited, the analyzing system is controlled so that a predetermined number of peaks will be sequentially selected as the precursor ion in descending order of their strengths. In another commonly known method, all the peaks, without limiting the number of peaks, having strengths equal to or larger than a predetermined threshold are selected as precursor ions.
These methods seem to entirely rely on the assumption that an ion having a higher peak strength ensures a higher identification probability. Although this assumption is not qualitatively wrong, it should be noted that the peak strength does not always correspond to the value of identification probability. For example, suppose that there are multiple peaks that can be chosen as a precursor ion. In some cases, choosing any one of these peaks will result in successful identification with high probability, while in other cases successful identification can be expected only when a specific peak among them is chosen. Such a difference cannot be quantitatively determined in advance (i.e. before the MS2 analysis is performed). This is one of the reasons that deteriorate the efficiency of the comprehensive identification.